(1) Field of the Invention
The present invention relates to a multi-cellular cellulose particle having a novel structure, and a process for the preparation thereof. More particularly, the present invention relates to a multi-cellular cellulose particle having a structure suitable for use as a carrier for a catalyst, enzyme or medicine, a starting material for an ion exchange member or adsorbing material, or a micro-carrier for culturing cells, and a process for the preparation of this multi-cellular cellulose particle.
(2) Description of the Related Art
Fine cellulose particles are widely utilized as packing materials for gel permeation chromatography (GPC). Moreover, since various functional groups can be easily introduced in the particles, they have a wide range of applications as ion exchange members and affinity chromatography bases. Recent developments in biochemistry and genetic engineering have caused the demand for these particles in the field of separation and purification of trace proteins in the living body to be greatly increased. Porous particles are included in present commercially available cellulose particles, and in most of these porous particles, a pore structure having a very fine pore size is produced for adjusting the exclusion limit molecular weight in GPC or adjusting the density of the particles, with the pore diameter is about 1 .mu.m at its largest.
For example, according to the process disclosed in U.S. Pat. No. 3,597,350, cellulose is dissolved at a concentration of 1 to 12% in an aqueous cuprammonium solution or the like, the cellulose solution is dispersed in benzene containing an emulsifier, and the dispersion is thrown into a regenerating bath to obtain cellulose microspheres. It is stated that the cellulose particles obtained according to this process have a cellulose density of 2 to 25% (weight/volume) and a pore diameter ranging from 2 to 2,000 .mu.m. In order to increase the pore diameter, the cellulose density must be reduced, resulting in a lowering of the mechanical strength of the particles. Therefore, porous particles having a pore diameter of at least 2 .mu.m, strong enough for ordinary use, cannot be practically obtained.
According to the process disclosed in U.S. Pat. No. 4,055,510, a suspension of viscose in a water-immiscible dispersion medium is heated at a temperature of 30.degree. to 100.degree. C. with continuous stirring to effect solidification, and the formed particles are decomposed by an acid to obtain spherical cellulose particles. But, the particles obtained by this process are hard and gelatinous and the pore diameter is still in the order of submicrons.
A cellulose sponge is known as a cellulose structure having a large pore diameter, wherein the pore diameter is larger than several hundred .mu.m and holes of a unit of several mm are opened.
A particulate cellulose sponge having a small pore diameter is not known. For production of a cellulose sponge, a process is usually adopted in which a large quantity of a crystal of finely divided sodium sulfate decahydrate is incorporated in viscose in advance, the viscose is cast in a mold and solidified by heating, and then the crystal of the Glauber salt is removed by water washing to form a porous structure (see, for example, Japanese Examined Pat. Publication No. 18-1580). A porous structure is usually formed according to a process in which a pore-forming material that can be removed afterward, such as Glauber salt, is incorporated in a solvent in advance. If it is intended to obtain a multi-cellular cellulose particle having a large pore diameter by combining this process with the above-mentioned process for the production of spherical particles, since a large amount of a pore-forming material having a controlled particle size is incorporated in the solution, the flowability of the solution is reduced and it is very difficult to form fine liquid drops having a uniform size.
Moreover, in the case of a particulate pore-forming material, sometimes the solubility of cellulose is reduced at the stage where the pore-forming material is incorporated, and thus a partial precipitation occurs. Furthermore, since a large excess of the pore-forming material must be incorporated in the cellulose solution, to greatly increase the void ratio in the interior of the particle or form a continuous hole structure, the mechanical strength is inevitably drastically reduced in the porous particle remaining after removal of the pore-forming material. In the cellulose sponge, a certain strength is attained by incorporating a fiber such as flax as a reinforcer into viscose in advance. According to this process, because of entanglement of the fiber in the cellulose solution, it is practically impossible to form uniform fine drops of the solution.
As is apparent from the foregoing description, it is very difficult to form particles by directly employing the process for the preparation of a cellulose sponge.
To summarize, a porous cellulose particle having many cells having a maximum inscribed sphere diameter of at least about 2 .mu.m, in which these cells form a continuous hole structure, has not been heretofore developed.
To obtain a high-water-content shaped article of a gel of a water-soluble polymer other than cellulose (this product is also known as "a porous article", but the porosity referred to in this case is based on the concept of very fine spaces in the network of molecules of the gel and is quite different from the concept of the cells of the present invention), a solution of the polymer is cast in a mold frame or formed into a coating film, the solution is frozen, and the frozen solution is vacuum-dried without thawing. It has been proposed that a gel shaped article is obtained by utilizing the conventional technique of fixing a gel structure of the solution by the freeze-vacuum-drying method (a process using polyvinyl alcohol is disclosed in Japanese Unexamined Pat. Publication No. 57-130543 and Japanese Unexamined Pat. Publication No. 57-159826, and a process using solubilized collagen is disclosed in Japanese Unexamined Pat. Publication No. 56-23,896). But this technique has a different object and procedure than that of the present invention.
As one mode of application of the multicellular cellulose particle, there can be mentioned a mode in which the particle is packed as a carrier in a column. In this case, if pores of the particle are small, it takes a long time for a mother liquid to pass through the particle, and accordingly, to improve the reaction efficiency in the column, the liquid pressure is elevated while reducing the size of the particle. But, if this technique is adopted, the flow resistance is increased and the particle is deformed. Thus, this technique is limited. A multi-cellular particle having a large pore diameter and a small flow resistance is especially suitable as a carrier for the separation and purification of a high-molecular-weight protein derived from a living body, and thus the development of a multi-cellular cellulose particle having a large pore diameter is desired.
In a micro-carrier used for the mass culturing of adherent cells, a method has been adopted in which the cultured cell concentration is increased to 10.sup.6 cell/ml by sticking cells to the surface of the particle. If a multi-cellular particle has a large pore diameter such that cells can enter into the interior of the particle and can adhere thereto, by holding the cells in the interior of the particle, the problem of cells falling from the surface caused by impingement among the micro-carrier particles can be solved, and a micro-carrier can be provided in which the cultured cell concentration is further improved by a dramatic increase of the effective adhesion surface area.
Porous cellulose particles having a pore diameter smaller than 2 .mu.m are mainly produced according to the conventional technique, cellulose particles having uniformly opened cells having a diameter of at least 2 .mu.m cannot be obtained according to this conventional technique.